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Background  Lignocellulosic materials provide an attractive replacement for food-based crops used to produce ethanol. Understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. In this study, eucalyptus down-regulated in expression of cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) or  p -coumaroyl quinate/shikimate 3′-hydroxylase (C3′H, EC 1.14.13.36) were evaluated for cell wall composition and reduced recalcitrance.    Results  Eucalyptus trees with down-regulated  C4H  or  C3′H  expression displayed lowered overall lignin content. The control samples had an average of 29.6 %, the  C3′H  reduced lines had an average of 21.7 %, and the  C4H  reduced lines had an average of 18.9 % lignin from wet chemical analysis. The  C3′H  and  C4H  down-regulated lines had different lignin compositions with average S/G/H ratios of 48.5/33.2/18.3 for the  C3′H  reduced lines and 59.0/39.8/1.2 for the  C4H  reduced lines, compared to the control with 65.9/33.2/1.0. Both the  C4H  and  C3′H  down-regulated lines had reduced recalcitrance as indicated by increased sugar release as determined using enzymatic conversion assays utilizing both no pretreatment and a hot water pretreatment.    Conclusions  Lowering lignin content rather than altering sinapyl alcohol/coniferyl alcohol/4-coumaryl alcohol ratios was found to have the largest impact on reducing recalcitrance of the transgenic eucalyptus variants. The development of lower recalcitrance trees opens up the possibility of using alternative pretreatment strategies in biomass conversion processes that can reduce processing costs.

biotechnology for biofuels

Down-regulation of p-coumaroyl quinate/shikimate 3′-hydroxylase (C3′H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla × E. grandis leads to improved sugar release