Downregulation of Cinnamyl Alcohol Dehydrogenase (CAD) Leads to Improved Saccharification Efficiency in Switchgrass

Abstract

The bioconversion of carbohydrates in the herbaceous bioenergy crop, switchgrass (Panicum virgatum L.), is limited by the associated lignins in the biomass. The cinnamyl alcohol dehydrogenase (CAD) gene encodes a key enzyme which catalyzes the last step of lignin monomer biosynthesis. Transgenic switchgrass plants were produced with a CAD RNAi gene construct under the control of the maize ubiquitin promoter. The transgenic lines showed reduced CAD expression levels, reduced enzyme activities, reduced lignin content, and altered lignin composition. The modification of lignin biosynthesis resulted in improved sugar release and forage digestibility. Significant increases of saccharification efficiency were obtained in most of the transgenic lines with or without acid pretreatment. A negative correlation between lignin content and sugar release was found among these transgenic switchgrass lines. The transgenic materials have the potential to allow for improved efficiency of cellulosic ethanol production.

Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population

“…Forward genetic screens have also led to the discovery of visual and lignin-related phenotypes, such as brown-midrib maize (Zea mays) and sorghum (Sorghum bicolor), which show improved digestion by ruminant animals and increased sugar yield by enzyme hydrolysis (Cherney et al, 1991;Marita et al, 2003;Vermerris et al, 2007). However, reducing lignin content either in mutant or transgenic lines can result in reduced biomass yields (Li and Chapple, 2010;Simmons et al, 2010;Fu et al, 2011aFu et al, , 2011b. This strategy is also undesirable for pyrolytic or chemical catalytic conversion processes that may utilize lignin for hydrocarbon fuels and aromatic coproducts (Venkatakrishnan et al, 2014).…”

“…In fact, in Arabidopsis, the impacts of genetic modifications of lignin are sometimes unseen without pretreatment (Li and Chapple, 2010). In switchgrass (Panicum virgatum), acid pretreatment increased saccharification yield in transgenic lines with reduced cinnamyl alcohol dehydrogenase (Fu et al, 2011b). Thermochemical pretreatments of biomass such as steam or ammonia explosion that reach temperatures above the range for lignin phase transition cause lignin to coalesce into molten bodies that redistribute within the biomass (Donohoe et al, 2008).…”

Molecular Breeding of Grasses by Transgenic Approaches for Biofuel Production

“…In the first half of 2011, three reports were published on the downregulation of lignin biosynthesis genes using RNAi technology to improve ethanol production from lignocellulosic biomass (Fu et al, 2011a;Fu et al, 2011b;Saathoff et al, 2011). The first two reports were published at almost the same time and both targeted the gene encoding cinnamyl alcohol dehydrogenase (CAD), a key enzyme for catalyzing the last step of lignin monomer biosynthesis.…”

“…The first two reports were published at almost the same time and both targeted the gene encoding cinnamyl alcohol dehydrogenase (CAD), a key enzyme for catalyzing the last step of lignin monomer biosynthesis. The resulting transgenic plants showed significantly fewer transcripts of the target gene, reduced CAD activity, lower lignin content (Fu et al, 2011b;Saathoff et al, 2011), and altered lignin composition (Fu et al, 2011b). Furthermore, these modified lignin biosynthesizers had improved sugar release from cell walls with or without chemical pre-treatment (Fu et al, 2011b;Saathoff et al, 2011).…”

“…The resulting transgenic plants showed significantly fewer transcripts of the target gene, reduced CAD activity, lower lignin content (Fu et al, 2011b;Saathoff et al, 2011), and altered lignin composition (Fu et al, 2011b). Furthermore, these modified lignin biosynthesizers had improved sugar release from cell walls with or without chemical pre-treatment (Fu et al, 2011b;Saathoff et al, 2011). In another study, the expression of the lignin biosynthesis-related O-methyltransferase gene was similarly downregulated, and the resultant transgenic plants had reduced lignin content, digestibility by cellulase, and up to 38% more efficient ethanol yield using conventional biomass fermentation processes (Fu et al, 2011a).…”