Dominant Suppression of Xylan Biosynthesis Using IRX10

Andrew Brandon^1,2, Mi Yeon Lee¹, Henrik Vibe Scheller^1,2

¹Joint Bioenergy Institute and Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

²Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA

The β-(1,4)-linked xylose homopolymer xylan comprises 20-30% of the biomass of dicot plant species and nearly half the biomass of grasses. It is the most abundant polysaccharide after cellulose in most plants and, thus, one of the most abundant biopolymers on earth. While its role in the cell wall is not entirely known, it is believed to coat and/or crosslink cellulose microfibrils. Mutants in xylan biosynthetic genes have thinner and weaker cell walls. The vessels of these mutants tend to collapse under the negative pressure of water transport, causing a severely dwarfed whole-plant phenotype. Despite its abundance in the cell wall and importance to plant health, the composition of xylan makes it unfavorable for the conversion of plant biomass to biofuels and bioproducts. The xylan backbone is composed entirely of the 5-carbon sugar xylose. Microbial fermentation of 5-carbon sugars is relatively inefficient, and the presence of 5-carbon sugars inhibits the fermentation of abundant 6-carbon sugars like glucose. Additionally, the xylan backbone is heavily substituted with acetyl groups. The released acetate creates a toxic environment for microbial fermentation. So, any engineering efforts that can reduce the amount of xylan in plant biomass can have significant positive impacts on the viability and cost-efficiency of advanced biofuels. Transformation of plants with dominant alleles is an ideal method to engineer a range of bioenergy crops, especially since few, if any, xylan mutants of biotechnologically relevant crops exist. We have therefore explored the possibility of developing dominant supressors of xylan biosynthesis. While xylan biosynthesis is still poorly understood, one pair of homologous enzymes Irregular xylem (IRX) 10 and 10-like has unambiguously demonstrated xylan synthase activity. We have been able to dramatically reduce or abolish this catalytic activity by identifying and mutating highly conserved residues in the IRX10 amino acid sequence. Then, we were able to out-compete the native IRX10 enzyme in a wild-type background by constitutively overexpressing the mutant form, the effect being to suppress xylan biosynthesis and reduce the amount of xylan in the plant to varying degrees.