title: Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility.
abstract: BACKGROUND
  Alfalfa (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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  Modifying 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.
authors.forename: Ajay
authors.initials: A
authors.lastname: Badhan
authors.forename: Long
authors.initials: L
authors.lastname: Jin
authors.forename: Yuxi
authors.initials: Y
authors.lastname: Wang
authors.forename: Shuyou
authors.initials: S
authors.lastname: Han
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kowalczys
authors.forename: Daniel Cw
authors.initials: DC
authors.lastname: Brown
authors.forename: Carlos Juarez
authors.initials: CJ
authors.lastname: Ayala
authors.forename: Marysia
authors.initials: M
authors.lastname: Latoszek-Green
authors.forename: Brian
authors.initials: B
authors.lastname: Miki
authors.forename: Adrian
authors.initials: A
authors.lastname: Tsang
authors.affiliation: Agriculture and Agri-Food Canada, Lethbridge Research Centre, 5403 1st Avenue South, Lethbridge, AB T1J 4B1, Canada. tim.mcallister@agr.gc.ca.
authors.forename: Tim
authors.initials: T
authors.lastname: McAllister