Reducing Xylan and Improving Lignocellulosic Biomass through Antimorphic and Heterologous Enzyme Expression

Large-scale, sustainable production of lignocellulosic bioenergy from plant biomass will likely depend on a variety of dedicated bioenergy crops. Compared to their genetic and genomic diversity, polysaccharide composition varies little from one species to the next. Genetic engineering can be used to dramatically improve plant biomass for biofuel production. Engineering strategies that act independently of genetic context are particularly important, since they can improve the polysaccharide composition in a variety of species. Cellulose is the most abundant polysaccharide in all plants and the source of the six-carbon sugar glucose that is, ultimately, converted by genetically engineered microbes into liquid fuels and a variety of useful commodity chemicals. The ideal bioenergy crop would be as cellulose-dense as possible, and overexpression of plant and fungal enzymes can dramatically increase cellulose production and make simpler to break down. The second most abundant polysaccharide in bioenergy crops is xylan, composed of the five-carbon sugar xylose. Xylan associates tightly with cellulose microfibrils and, via its side chains, forms covalent linkages with other components of the cell wall, making xylan a significant contributor to the difficulty of biomass deconstruction. Xylan degrades and releases toxic compounds during the pretreatment of lignocellulosic biomass. These compounds and xylose itself are significantly detrimental to the microbial fermentation of glucose. Thus, a reduction in the amount of xylan and its complexity are additional characteristics that should be engineered into the ideal bioenergy crop. From a plant genetic engineering perspective, reducing the biosynthesis of a polysaccharide is significantly more challenging than increasing it, as a loss-of-function mutation in a specific gene would typically be required. Bioenergy crops often have multiple, functionally redundant, copies of a gene in their genome. Even with the advancement of genome editing technologies like CRISPR, homologous enzymes may have diverged in DNA sequence, making the task of knocking out each copy very laborious and time-consuming. I developed a protein-level genetic engineering approach to significantly reduce xylan content by overexpressing a catalytically dead or diminished (i.e. antimorphic) version of the xylan biosynthetic enzyme Irregular xylem10 (IRX10) in the model plant Arabidopsis thaliana. This work additionally provides experimental evidence supporting the hypothesis that xylan biosynthesis requires a complex of proteins in the Golgi, a complex in which IRX10 is the agent of catalysis. The journal publication of this work has been adapted in Chapter 1. High-level expression of the antimorph “out-competed” the native enzyme for its place in the complex, thereby generating xylan synthase complexes with little to no catalytic activity. A major advantage to this strategy is that sufficiently high expression would likely have the same effect on any redundant IRX10 homologs the plant may express. Additionally, the amino acid residues mutated to create the antimorphic protein are fully conserved in all land plants, so the technology could be translated to a variety of bioenergy crops with relative ease and the US Patent Application regarding it is the subject of Chapter 2. Continuing my study of dominant genetic engineering strategies for improving the qualities of biomass crops, I conducted an intensive study of carbohydrate active enzymes and proteins that have been heterologously expressed in plants to modify polysaccharides in muro. This invited review, for future publication in Frontiers in Plant Science, is covered in Chapter 3.

Advisor: Scheller, Henrik V.