Genetic Modification Of Lignocellulose Recalcitrance For Efficient Biomass Enzymatic Saccharification By Editing OsCOMT And OsCAD2 Genes In Rice

Lignocellulose represents the most abundant renewable biomass resource for biofuels and biochemicals on the earth.However,lignocellulose recalcitrance decides a costly biomass process unacceptable for bioethanol production at large scale.Lignocellulose recalcitrance is in principle determined by plant cell wall composition,wall polymer characteristics,and wall network styles.As a major wall polymer,lignin deposition largely contributes to lignocellulose recalcitrance,and particularly lignin monomer proportions distinctively affect biomass enzymatic saccharification in bioenergy crops examined.Despite that genetic engineering of lignin biosynthesis has been implemented to reduce lignocellulose recalcitrance,it simply affects plant growth and biomass yield.More importantly,it remains to explore how lignin modification could enhance biomass enzymatic saccharification.In this study,therefore,we initially identified two candidate genes involved in lignin biosynthesis,and selected their mutants using CRISPR/Cas9 editing technology.We then characterized lignin and other polymer features,and finally proposed a mechanism model to elucidate why biomass enzymatic saccharification was largely enhanced for high bioethanol production in the rice mutants.The main results were described below:1.Identification of OsCOMT and OsCAD2 as desirable genes for lignin modification This study screened out 11 candidate genes from total 90 lignin biosynthesis genes according to the CREP data and gene chips in four rice fragile-culm mutants.Correlation analyses showed that OsCOMT,OsCAD2,OsCCR2,OsC3H,OsPAL8 and OsPAL2 would be the candidate genes for genetic engineering of lignin biosynthesis.Using CRISPR/Cas9 technology,we generated those genes mutants,and identified the OsCOMT and OsCAD2 mutants as the desirable materials for following experiments.2.Improved lignocellulose recalcitrance in the OsCOMT and OsCAD2 mutantsUsing multiple advanced approaches such as nitrobenzene oxidation（NBO）,nuclear magnetic resonance（NMR）and gel permeation chromatographic（GPC）,this study characterized that the Oscomt and Oscad2 mutants were of reduced lignin levels by 15%and 16%,with molecular weight（Mw）reduced by 36%and 24%,compared to the wild type（NPB）.In particular,the Oscomt mutant showed lignin G-monomer increased by 44%and S-monomer reduced by 48%,leading to its S/G ratio decreased by 71%.By comparison,the Oscad2 mutant had H-monomer and S/G reduced by 33.4%and 14%,respectively.Meanwhile,the Oscomt and Oscad2 mutants had cellulose and hemicelluloses levels increased by 20%and 30%,which may compensate for their lignin reduction.In addition,two mutants did not show much affected plant growth and biomass yields.3.Enhanced biomass saccharification in the Oscomt and Oscad2 mutantsUnder mild alkali（1%Na OH）pretreatments,two mutants showed biomass enzymatic saccharification enhanced by 65%and 72%,compared to the wild type.Particularly,the Oscomt mutant was of relatively high lignin-carbohydrate complex,interpreting why the mild alkali pretreatment could cause more lignin extraction at 26%-38%and much higher biomass porosity by 77%for remarkably enhanced biomass enzymatic saccharification.As a comparison,the Oscad2 mutant showed 70%lignin extraction from alkali pretreatment with biomass porosity raised by 58%-87%,which led to much less lignin adsorption with cellulases for high cellulose accessibility.4.OsCAD2 and OsCOMT co-expression for integrated enhancements of biomass yield and saccharificationBy performing genetic crossing between Oscad2 and Oscomt mutants,this study selected their hybridized generations（COMT-OX/cad,comt/cad）with much raised biomass and grain yields.As a comparison,both COMT-OX/cad and comt/cad straws were of lignin levels reduced by 26%and 32%relative to their parents.Notably,both straw samples also showed a complete biomass enzymatic saccharification from mild alkali pretreatments performed in this study.5.OsCOMT and OsCAD2 residues appliable for active biosorbentUsing the lignin-rich residues obtained from biomass enzymatic hydrolysis after alkali pretreatment,this study examined their adsorption with Cd²⁺and found that the Oscomt mutant had increased adsorption capacity by 10%.With respect to its lignin rich at G-monomer,it suggests that the Oscomt mutant could be considered to generate vanillin through oxidative depolymerization with lignin,which would be applicable to produce vanilla flavors and flavorings.In addition,due to reduced lignin molecular weights,two mutants may be of advantage for nano-lignin production in the future.In conclusion,genetic editing of OsCOMT and OsCAD2 genes could not only maintain plant growth and biomass yields,but also remarkably improve lignocellulose recalcitrance in their genetic mutants,leading to a mechanism model proposed to highlight why the Oscomt and Oscad2 mutants were of remarkably enhanced biomass enzymatic saccharification.Therefore,this study has demonstrated a genetic approach applicable for cell wall modification in bioenergy crops,providing a cost-effective and green-like strategy for biomass utilization.