Comparison of synthetic and post-synthetic molecular genetic methods of reducing xylan glucuronidation in hybrid aspen for biorefinery applications

BACKGROUND: The pattern of xylan backbone substitution by 4-O-methylglucuronic acid (mGlcA) and O-acetyl groups generated during biosynthesis in the Golgi apparatus plays a key role in molecular interactions between secondary cell wall polymers in hardwoods, thus regulating biorefinery properties of woody biomass. This study aims at comparing the effects of two different approaches to reduce xylan glucuronidation, targeting either xylan biosynthesis by RNAi suppression of GLUCURONIC SUBSTITUTION OF XYLAN1 genes (denoted Gux1RNAi) or by xylan post-synthetic modification in the secondary cell wall by apoplastic wood-specific expression of a fungal α-glucuronidase from family GH67 (denoted WP:GH67) in hybrid aspen (Populus tremula L. x tremuloides Michx.). The effects of reducing xylan glucuronidation by these two approaches on xylan acetylation and solubility, and recalcitrance of wood to enzymatic hydrolysis were compared. RESULTS: Both approaches reduced mGlcA/xylose (Xyl) ratio by approx. 30% without altering Xyl content in alkali extracts, while xylan acetyl content in subcritical water extracts was increased only in Gux1RNAi lines. The Gux1RNAi lines showed reduced glucuronidation in the major xylan domain containing evenly-spaced mGlcA, whereas the WP:GH67 lines were affected in both odd and even glucuronidation patterns. Interestingly, the arrays containing mGlcA every second xylosyl unit were not affected by any modification, whereas an increase in xylo-oligosaccharides containing O-2,3 diacetylated Xyl was found in Gux1RNAi lines only. Neither modification consistently affected glucose or Xyl yields in saccharification without or with acid pretreatment, whereas xylan extractability in subcritical water was increased in Gux1RNAi, but not in WP:GH67 lines. CONCLUSION: Our study supports the hypothesis that acetylation competes with glucuronidation by GUX1 during biosynthesis, and increased acetylation caused by GUX1 suppression rather than reduced xylan glucuronidation per se improves xylan extractability. The 30% reduction in glucuronidation did not affect enzymatic saccharification, supporting the concept that cell wall structure and recalcitrance in transgenic lines are maintained by xylan domains which were not affected by either transgenic modification.