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Title: Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis

Abstract

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. In this paper, we engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Finally, decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Authors:
 [1]; ORCiD logo [1]; ORCiD logo [2];  [3];  [2]; ORCiD logo [2];  [4];  [3];  [5];  [4];  [4];  [6];  [6];  [6];  [1]; ORCiD logo [7];  [1];  [1];  [8];  [8] more »;  [8];  [8];  [8];  [9];  [9]; ORCiD logo [8];  [1];  [7];  [7];  [7];  [7]; ORCiD logo [7]; ORCiD logo [10];  [8];  [4];  [8]; ORCiD logo [7];  [4];  [6];  [11];  [3];  [1] « less
  1. Univ. of Georgia, Athens, GA (United States). Complex Carbohydrate Research Center; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). DOE-BioEnergy Science Center (BESC)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). DOE-BioEnergy Science Center (BESC). Biosciences Division; UT-ORNL Joint Inst. for Biological Sciences, Oak Ridge, TN (United States)
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). DOE-BioEnergy Science Center (BESC); Univ. of Tennessee, Knoxville, TN (United States). Dept. of Plant Sciences
  4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). DOE-BioEnergy Science Center (BESC); Noble Research Inst., Ardmore, OK (United States)
  5. Univ. of Georgia, Athens, GA (United States). Complex Carbohydrate Research Center
  6. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). DOE-BioEnergy Science Center (BESC); Univ. of Georgia, Athens, GA (United States). Inst. of Plant Breeding, Genetics and Genomics
  7. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). DOE-BioEnergy Science Center (BESC); National Renewable Energy Lab. (NREL), Golden, CO (United States)
  8. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). DOE-BioEnergy Science Center (BESC). Biosciences Division
  9. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). DOE-BioEnergy Science Center (BESC); ArborGen, Inc., Ridgeville, SC (United States)
  10. Univ. of Georgia, Athens, GA (United States). Complex Carbohydrate Research Center. Dept. of Plant Biology; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). DOE-BioEnergy Science Center (BESC)
  11. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). DOE-BioEnergy Science Center (BESC). Biosciences Division; UT-ORNL Joint Inst. for Biological Sciences, Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States). Dept. of Chemical and Biomolecular Engineering. Dept. of Forestry, Wildlife, and Fisheries
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Georgia, Athens, GA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); National Science Foundation (NSF)
OSTI Identifier:
1460184
Grant/Contract Number:  
AC05-00OR22725; SC0015662; DBI-0421683; IOS-0923992
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nature Biotechnology
Additional Journal Information:
Journal Volume: 36; Journal Issue: 3; Journal ID: ISSN 1087-0156
Publisher:
Springer Nature
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; biofuels; field trials; molecular engineering in plants

Citation Formats

Biswal, Ajaya K., Atmodjo, Melani A., Li, Mi, Baxter, Holly L., Yoo, Chang Geun, Pu, Yunqiao, Lee, Yi-Ching, Mazarei, Mitra, Black, Ian M., Zhang, Ji-Yi, Ramanna, Hema, Bray, Adam L., King, Zachary R., LaFayette, Peter R., Pattathil, Sivakumar, Donohoe, Bryon S., Mohanty, Sushree S., Ryno, David, Yee, Kelsey, Thompson, Olivia A., Rodriguez, Miguel, Dumitrache, Alexandru, Natzke, Jace, Winkeler, Kim, Collins, Cassandra, Yang, Xiaohan, Tan, Li, Sykes, Robert W., Gjersing, Erica L., Ziebell, Angela, Turner, Geoffrey B., Decker, Stephen R., Hahn, Michael G., Davison, Brian H., Udvardi, Michael K., Mielenz, Jonathan R., Davis, Mark F., Nelson, Richard S., Parrott, Wayne A., Ragauskas, Arthur J., Neal Stewart, C., and Mohnen, Debra. Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis. United States: N. p., 2018. Web. doi:10.1038/nbt.4067.
Biswal, Ajaya K., Atmodjo, Melani A., Li, Mi, Baxter, Holly L., Yoo, Chang Geun, Pu, Yunqiao, Lee, Yi-Ching, Mazarei, Mitra, Black, Ian M., Zhang, Ji-Yi, Ramanna, Hema, Bray, Adam L., King, Zachary R., LaFayette, Peter R., Pattathil, Sivakumar, Donohoe, Bryon S., Mohanty, Sushree S., Ryno, David, Yee, Kelsey, Thompson, Olivia A., Rodriguez, Miguel, Dumitrache, Alexandru, Natzke, Jace, Winkeler, Kim, Collins, Cassandra, Yang, Xiaohan, Tan, Li, Sykes, Robert W., Gjersing, Erica L., Ziebell, Angela, Turner, Geoffrey B., Decker, Stephen R., Hahn, Michael G., Davison, Brian H., Udvardi, Michael K., Mielenz, Jonathan R., Davis, Mark F., Nelson, Richard S., Parrott, Wayne A., Ragauskas, Arthur J., Neal Stewart, C., & Mohnen, Debra. Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis. United States. doi:10.1038/nbt.4067.
Biswal, Ajaya K., Atmodjo, Melani A., Li, Mi, Baxter, Holly L., Yoo, Chang Geun, Pu, Yunqiao, Lee, Yi-Ching, Mazarei, Mitra, Black, Ian M., Zhang, Ji-Yi, Ramanna, Hema, Bray, Adam L., King, Zachary R., LaFayette, Peter R., Pattathil, Sivakumar, Donohoe, Bryon S., Mohanty, Sushree S., Ryno, David, Yee, Kelsey, Thompson, Olivia A., Rodriguez, Miguel, Dumitrache, Alexandru, Natzke, Jace, Winkeler, Kim, Collins, Cassandra, Yang, Xiaohan, Tan, Li, Sykes, Robert W., Gjersing, Erica L., Ziebell, Angela, Turner, Geoffrey B., Decker, Stephen R., Hahn, Michael G., Davison, Brian H., Udvardi, Michael K., Mielenz, Jonathan R., Davis, Mark F., Nelson, Richard S., Parrott, Wayne A., Ragauskas, Arthur J., Neal Stewart, C., and Mohnen, Debra. Mon . "Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis". United States. doi:10.1038/nbt.4067. https://www.osti.gov/servlets/purl/1460184.
@article{osti_1460184,
title = {Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis},
author = {Biswal, Ajaya K. and Atmodjo, Melani A. and Li, Mi and Baxter, Holly L. and Yoo, Chang Geun and Pu, Yunqiao and Lee, Yi-Ching and Mazarei, Mitra and Black, Ian M. and Zhang, Ji-Yi and Ramanna, Hema and Bray, Adam L. and King, Zachary R. and LaFayette, Peter R. and Pattathil, Sivakumar and Donohoe, Bryon S. and Mohanty, Sushree S. and Ryno, David and Yee, Kelsey and Thompson, Olivia A. and Rodriguez, Miguel and Dumitrache, Alexandru and Natzke, Jace and Winkeler, Kim and Collins, Cassandra and Yang, Xiaohan and Tan, Li and Sykes, Robert W. and Gjersing, Erica L. and Ziebell, Angela and Turner, Geoffrey B. and Decker, Stephen R. and Hahn, Michael G. and Davison, Brian H. and Udvardi, Michael K. and Mielenz, Jonathan R. and Davis, Mark F. and Nelson, Richard S. and Parrott, Wayne A. and Ragauskas, Arthur J. and Neal Stewart, C. and Mohnen, Debra},
abstractNote = {Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. In this paper, we engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Finally, decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.},
doi = {10.1038/nbt.4067},
journal = {Nature Biotechnology},
issn = {1087-0156},
number = 3,
volume = 36,
place = {United States},
year = {2018},
month = {2}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Figures / Tables:

Fig. 1. Fig. 1.: Saccharification yield from switchgrass, rice, and poplar GAUT4-KD lines and bioconversion of switchgrass GAUT4-KD to ethanol. (a-i) Glucose, xylose, and total sugar release, respectively, from greenhouse-grown PvGAUT4KD (a-c), OsGAUT4-KD (d-f), and PdGAUT4-KD (g-i) lines compared to controls. Materials used were three-month-old switchgrass tillers, three-month-old rice aerial biomass, andmore » ninemonth-old poplar wood. (j and k) Ethanol yield from yeast-based separate hydrolysis and fermentation (SHF) of hot water-pretreated greenhouse-grown switchgrass PvGAUT4-KD lines 2A, 2B and 4A and wild type (WT) biomass, shown as (j) mg EtOH/g cellulose and (k) mg EtOH/g dry biomass. Biomass hydrolyses were carried out with commercial enzyme blends (Ctec2, Htec2, and 188; Novozymes) at 50°C, pH 4.5 for 5 days; fermentation with S. cerevisiae D5A at 35°C, pH 4.5, 150 rpm. Switchgrass caffeic acid 3-O-methyltransferase knockdown transgenic (COMT3-TG) and corresponding WT lines (COMT3-WT) were included as a reference21. (l) Time course fermentation of whole biomass in (k) measured as weight loss of fermentation bottles. Fermentation was performed with three technical replicates of two independently grown biomass samples of three genetically identical clones of a single transgenic event (n = 6). (m-o) Glucose, xylose, and total sugar release, respectively, from field-grown PvGAUT4-KD compared to controls in the first (2013), second (2014), and third (2015) growing seasons. (p) Ethanol yield from yeast-based separate hydrolysis and fermentation (SHF) of hot water-pretreated field-grown year 3 switchgrass PvGAUT4-KD and WT biomass, shown as grams EtOH/plant. For greenhouse-grown biomass: WT and PvGAUT4-KD lines, n = 4; WT and OsGAUT4-KD lines, n = 6; WT P. deltoides, n = 25; PdGAUT4-KD lines, n = 15. For fieldgrown biomass PvGAUT4-KD and WT: n = 3. Data are presented as box plots showing the median as well as the 25th and 75th percentiles. Ends of whiskers are set at 1.5*IQR above and below the third and first quartiles, respectively. Statistical analysis was done using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test for greenhousegrown biomass, or by Fisher’s least significant difference method for field-grown biomass (*$P$ < 0.05, **$P$ < 0.001).« less

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    Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.