Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility

Abstract

BackgroundAlfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production.ResultsIn this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line.ConclusionModifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines.

MicroRNA156 as a promising tool for alfalfa improvement

“…Wild type (WT) alfalfa ( Medicago sativa ) clone N4.4.2 (Badhan et al ., ) was obtained from Dr. Daniel Brown (Agriculture and Agri‐Food Canada) and was used in transformation assays and all other aspects of this work. WT and transgenic alfalfa plants were maintained in a greenhouse kept at 21–23 °C, 16 h light per day (halogen lights were applied after 18:00 h), light intensity of 380–450 W/m 2 (approximately 500 W/m 2 at high noontime) and a relative humidity (RH) of 70%.…”

Diversity of fungal feruloyl esterases: updated phylogenetic classification, properties, and industrial applications

“…Different strategies have been applied to create FAE mutants which can tolerate the high temperatures in bioprocesses [118–120]. Furthermore, transgenic plants have been manipulated specifically for biofuel production to reduce recalcitrance of cell walls prior to saccharification, which also enhance the digestibility and biomass conversion for livestock (e.g., [32–35, 121]). Besides, FAEs are not only used for complete hydrolysis of lignocellulosic materials, but they can also be applied for manipulating the structure of oligosaccharides e.g., in production of xylo -oligosaccharides [122] which are industrially important functional food additives with prebiotic properties [123].…”

Expression of Glycosyl Hydrolases in Lignocellulosic Feedstock: An Alternative for Affordable Cellulosic Ethanol Production

“…The altered cell wall composition resulted in the release of more fermentable sugars after enzymatic hydrolysis [75]. Several studies on maize, tall fescue, and alfalfa expressing cellulolytic enzymes reported that transgenic plants have higher glucan conversion than non-transgenic plants after thermochemical pretreatment and enzymatic digestion [74,75,85,90,91]. These results indicate that expressing cell wall-degrading enzymes in biomass crops can have positive effects on saccharification efficiency, which allows for more ethanol production.…”

“…In vitro digestibility is enhanced in glycosyl hydrolase expressing feedstock to release more sugar compared to wild type [75,85]. However, few studies have reported processing transgenic feedstock using scalable technologies for pretreatment and saccharification, let alone measuring ethanol yield resultant from fermentation.…”