Genetic Modification Of Lignocellulose Structures For Enhanced Biomass Enzymatic Saccharification In The Transgenic Rice Plants Over-expressing Native Glycoside Hydrolases Genes

Lignocellulose represents enormous biomass resource for biofuels and chemical production.Importantly,cellulosic ethanol is an excellent additive to the petroleum for less net carbon and harmful gas release.In general,cellulosic ethanol process involves in three major steps:initial physical and chemical pretreatment for cell wall destruction,sequential enzymatic hydrolysis for soluble sugar release and final yeast fermentation for bioethanol production.However,due to lignocellulose recalcitrance,the current bioethanol conversion requires a strong pretreatment and expensive enzyme loading,leading to a costly bioethanol production with potential secondary pollution to the environment.Hence,genetic modification of plant cell walls is considered as a promising solution to the recalcitrance.Rice is a major food crop in China,providing large amounts of straw for biofuels,whereas Miscanthus as a native C4 grass,is a leading bioenergy crop with much high lignocellulose yield and well adaption to the environment.In this study,we selected dozen transgenic rice lines that respectively over-expressed OsGH9s and OsGH10s genes,and then examined their cell wall compositions and wall polymer features using our well-established approaches.Compared to the control cultivar(NPB),almost all OsGH9transgenic lines showed a normal plant growth with little changes of mechanical strength and biomass yields.However,both OsGH9B1 and OsGH9B3 transgenic plants showed significantly reduced cellulose features(DP,Cr I),which caused much enhanced biomass enzymatic saccharification with highest bioethanol yield obtained at 22.5%(%dry matter),even though under mild alkali pretreatment(0.5%NaOH,50 ~0C).Taken all together,the results suggested that OsGH9B1,B3,B16 enzymes may involve in post-modification of cellulose microfibrils in the transgenic rice plants.Meanwhile,this study performed a comparative and correlative analysis between transcription expression levels and hemicellulosic features in total 24 representative rice mutants.The results indicated that OsGH10-3 and OsGH10-4 may respectively involve in alkali-extractable and none-alkali-extractable hemicellulose modification in the transgenic rice lines.Unlike OsGH9B transgenic rice plants,both OsGH10-3 and OsGH10-4 transgenic plants showed significantly reduced plant heights and mechanical strength,but they had significantly increased plant lodging resistance,compared to the control cultivar.Using total 247 OsGH9s and OsGH10s transgenic rice lines,this study established a near infrared spectroscopy(NIRS)assay for major wall polymer features and biomass saccharification.Hence,this study generated ten equations for predicting biomass enzymatic saccharification and seven equations for major wall polymer features,with determination coefficient on calibration(R~2)at 0.75-0.98,cross-validation(R~2cv)at0.69-0.98,and the ratio performance deviation(RPD)at 1.79-6.63,suggesting a perfect prediction capacity for high-throughput screening of bioenergy crops.In addition,total199 Miscanthus accessions were determined with largely varied biomass enzymatic saccharification under different pretreatments,which was suitable for NIRS assay.Therefore,seven optimal equations were generated with high R~2/R~2cv/R~2ev and RPD values,indicating that they could be applicable for high-throughput screening of Miscanthus germplasm accessions.