title: Directed plant cell-wall accumulation of iron: embedding co-catalyst for efficient biomass conversion.
abstract: BACKGROUND
  Plant lignocellulosic biomass is an abundant, renewable feedstock for the production of biobased fuels and chemicals. Previously, we showed that iron can act as a co-catalyst to improve the deconstruction of lignocellulosic biomass. However, directly adding iron catalysts into biomass prior to pretreatment is diffusion limited, and increases the cost of biorefinery operations. Recently, we developed a new strategy for expressing iron-storage protein ferritin intracellularly to accumulate iron as a catalyst for the downstream deconstruction of lignocellulosic biomass. In this study, we extend this approach by fusing the heterologous ferritin gene with a signal peptide for secretion intoArabidopsiscell walls (referred to here as FerEX).
  
  
  RESULTS
  The transgenicArabidopsisplants. FerEX. accumulated iron under both normal and iron-fertilized growth conditions; under the latter (iron-fertilized) condition, FerEX transgenic plants showed an increase in plant height and dry weight by 12 and 18 %, respectively, compared with the empty vector control plants. The SDS- and native-PAGE separation of cell-wall protein extracts followed by Western blot analyses confirmed the extracellular expression of ferritin in FerEX plants. Meanwhile, Perls' Prussian blue staining and X-ray fluorescence microscopy (XFM) maps revealed iron depositions in both the secondary and compound middle lamellae cell-wall layers, as well as in some of the corner compound middle lamella in FerEX. Remarkably, their harvested biomasses showed enhanced pretreatability and digestibility, releasing, respectively, 21 % more glucose and 34 % more xylose than the empty vector control plants. These values are significantly higher than those of our recently obtained ferritin intracellularly expressed plants.
  
  
  CONCLUSIONS
  This study demonstrated that extracellular expression of ferritin inArabidopsiscan produce plants with increased growth and iron accumulation, and reduced thermal and enzymatic recalcitrance. The results are attributed to the intimate colocation of the iron co-catalyst and the cellulose and hemicellulose within the plant cell-wall region, supporting the genetic modification strategy for incorporating conversion catalysts into energy crops prior to harvesting or processing at the biorefinery.
authors: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors: Chien-Yuan
authors: CY
authors: Lin
authors: Forest Biopolymer Science and Engineering, USDA Forest Service, Forest Products Laboratory, Madison, WI 53726 USA.
authors: Joseph E
authors: JE
authors: Jakes
authors: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors: Bryon S
authors: BS
authors: Donohoe
authors: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors: Peter N
authors: PN
authors: Ciesielski
authors: Department of Biological Science, Purdue University, West Lafayette, IN 47907 USA.
authors: Haibing
authors: H
authors: Yang
authors: X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 USA.
authors: Sophie-Charlotte
authors: SC
authors: Gleber
authors: X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 USA.
authors: Stefan
authors: S
authors: Vogt
authors: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA ; Department of Plant Biology, Michigan State University, East Lansing, MI 48824 USA.
authors: Shi-You
authors: SY
authors: Ding
authors: Department of Environmental Science and Technology, University of Maryland, College Park, MD 20742 USA.
authors: Wendy A
authors: WA
authors: Peer
authors: Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742 USA.
authors: Angus S
authors: AS
authors: Murphy
authors: Department of Biological Science, Purdue University, West Lafayette, IN 47907 USA.
authors: Maureen C
authors: MC
authors: McCann
authors: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors: Michael E
authors: ME
authors: Himmel
authors: National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors: Melvin P
authors: MP
authors: Tucker
authors: Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
authors: Hui
authors: 0000-0002-3550-188X
authors: H
authors: Wei
keywords: Biomass; Ferritin; High-throughput hot-water pretreatment; Iron accumulation; Iron co-catalyst; Perls’ Prussian blue staining; Saccharification; Sugar release; Transgenic Arabidopsis; X-ray fluorescence microscopy