Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase

Significance In the transition from a fossil-based to a bio-based economy, bioethanol will be generated from the lignocellulosic biomass of second-generation biofuel crops, such as poplar. The lignin polymers in the plant cell walls represent the main factor determining the recalcitrance of biomass to enzymatic processing. We have grown genetically modified poplars, down-regulated for cinnamoyl-CoA reductase (CCR), an enzyme in the lignin biosynthetic pathway, in field trials in Belgium and France. We show that wood samples derived from the transgenic trees are more easily processed into ethanol. However, strong down-regulation also affected biomass yield. In conclusion, CCR down-regulation may become a successful strategy to improve biomass processing if the yield penalty can be overcome. Lignin is one of the main factors determining recalcitrance to enzymatic processing of lignocellulosic biomass. Poplars (Populus tremula x Populus alba) down-regulated for cinnamoyl-CoA reductase (CCR), the enzyme catalyzing the first step in the monolignol-specific branch of the lignin biosynthetic pathway, were grown in field trials in Belgium and France under short-rotation coppice culture. Wood samples were classified according to the intensity of the red xylem coloration typically associated with CCR down-regulation. Saccharification assays under different pretreatment conditions (none, two alkaline, and one acid pretreatment) and simultaneous saccharification and fermentation assays showed that wood from the most affected transgenic trees had up to 161% increased ethanol yield. Fermentations of combined material from the complete set of 20-mo-old CCR–down-regulated trees, including bark and less efficiently down-regulated trees, still yielded ∼20% more ethanol on a weight basis. However, strong down-regulation of CCR also affected biomass yield. We conclude that CCR down-regulation may become a successful strategy to improve biomass processing if the variability in down-regulation and the yield penalty can be overcome.

[1]  Mark T. Holtzapple,et al.  Simultaneous saccharification and fermentation of lime-treated biomass , 2001, Biotechnology Letters.

[2]  John Ralph,et al.  Identification of the structure and origin of a thioacidolysis marker compound for ferulic acid incorporation into angiosperm lignins (and an indicator for cinnamoyl CoA reductase deficiency). , 2007, The Plant journal : for cell and molecular biology.

[3]  Markus Pauly,et al.  Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin , 2010, Journal of visualized experiments : JoVE.

[4]  S. Balzergue,et al.  Characterization of a cinnamoyl-CoA reductase 1 (CCR1) mutant in maize: effects on lignification, fibre development, and global gene expression , 2011, Journal of experimental botany.

[5]  Paul Dupree,et al.  Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana , 2013, Biotechnology for Biofuels.

[6]  John Ralph,et al.  Metabolic engineering of novel lignin in biomass crops. , 2012, The New phytologist.

[7]  M. Auer,et al.  Engineering of plants with improved properties as biofuels feedstocks by vessel-specific complementation of xylan biosynthesis mutants , 2012, Biotechnology for Biofuels.

[8]  C. Chapple,et al.  The genetics of lignin biosynthesis: connecting genotype to phenotype. , 2010, Annual review of genetics.

[9]  M. Pauly,et al.  Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates , 2010, Journal of Visualized Experiments.

[10]  Gregory L Helms,et al.  The Arabidopsis cinnamoyl CoA reductase irx4 mutant has a delayed but coherent (normal) program of lignification. , 2006, The Plant journal : for cell and molecular biology.

[11]  Joshua S Yuan,et al.  Plants to power: bioenergy to fuel the future. , 2008, Trends in plant science.

[12]  Godelieve Gheysen,et al.  Transcription factor WRKY23 assists auxin distribution patterns during Arabidopsis root development through local control on flavonol biosynthesis , 2012, Proceedings of the National Academy of Sciences.

[13]  D. Dickmann.  Silviculture and biology of short-rotation woody crops in temperate regions: then and now. , 2006 .

[14]  B. Solomon,et al.  Biofuels and sustainability , 2010, Annals of the New York Academy of Sciences.

[15]  Matthias Fladung,et al.  The 20-year environmental safety record of GM trees , 2010, Nature Biotechnology.

[16]  F. Breusegem,et al.  Towards a carbon-negative sustainable bio-based economy , 2013, Front. Plant Sci..

[17]  Joachim Kopka,et al.  Molecular phenotyping of lignin-modified tobacco reveals associated changes in cell-wall metabolism, primary metabolism, stress metabolism and photorespiration. , 2007, The Plant journal : for cell and molecular biology.

[18]  L. Jouanin,et al.  Impact of CCR1 silencing on the assembly of lignified secondary walls in Arabidopsis thaliana. , 2009, The New phytologist.

[19]  Gilles Pilate,et al.  Field trials with lignin-modified transgenic trees. , 2012 .

[20]  B. Sundberg,et al.  Downregulation of Cinnamoyl-Coenzyme A Reductase in Poplar: Multiple-Level Phenotyping Reveals Effects on Cell Wall Polymer Metabolism and Structure[W] , 2007, The Plant Cell Online.

[21]  W. Boerjan,et al.  Lignin Biosynthesis and Structure , 2010 .

[22]  W. Boerjan,et al.  Lignin biosynthesis. , 2003, Annual review of plant biology.

[23]  Robert W. Sykes,et al.  Antisense Down-Regulation of 4CL Expression Alters Lignification, Tree Growth, and Saccharification Potential of Field-Grown Poplar1[W][OA] , 2010, Plant Physiology.

[24]  M. Holtzapple,et al.  Oxidative lime pretreatment of high-lignin biomass , 2001, Applied biochemistry and biotechnology.

[25]  Stephen Y. Lin,et al.  Methods in Lignin Chemistry , 1992, Springer Series in Wood Science.

[26]  C. Chapple,et al.  Can genetic engineering of lignin deposition be accomplished without an unacceptable yield penalty? , 2013, Current opinion in biotechnology.

[27]  L. Lynd,et al.  How biotech can transform biofuels , 2008, Nature Biotechnology.

[28]  F. Meinzer,et al.  Tyloses and Phenolic Deposits in Xylem Vessels Impede Water Transport in Low-Lignin Transgenic Poplars: A Study by Cryo-Fluorescence Microscopy1[W][OA] , 2010, Plant Physiology.

[29]  K. Kang,et al.  Designed for deconstruction--poplar trees altered in cell wall lignification improve the efficacy of bioethanol production. , 2012, The New phytologist.

[30]  J. Ralph,et al.  Syntheses of lignin-derived thioacidolysis monomers and their uses as quantitation standards. , 2012, Journal of agricultural and food chemistry.

[31]  M. Auer,et al.  Engineering secondary cell wall deposition in plants , 2012, Plant biotechnology journal.

[32]  R. Dixon,et al.  Improving Saccharification Efficiency of Alfalfa Stems Through Modification of the Terminal Stages of Monolignol Biosynthesis , 2008, BioEnergy Research.

[33]  M. Pauly,et al.  A High-Throughput Platform for Screening Milligram Quantities of Plant Biomass for Lignocellulose Digestibility , 2010, BioEnergy Research.

[34]  J. Ralph,et al.  Using the acetyl bromide assay to determine lignin concentrations in herbaceous plants: some cautionary notes. , 1999, Journal of agricultural and food chemistry.

[35]  Richard A Dixon,et al.  Lignin modification improves fermentable sugar yields for biofuel production , 2007, Nature Biotechnology.

[36]  Mark F. Davis,et al.  Lignin content in natural Populus variants affects sugar release , 2011, Proceedings of the National Academy of Sciences.

[37]  R. Custers.  First GM trial in Belgium since 2002 , 2009, Nature Biotechnology.

[38]  Bartel Vanholme,et al.  A Systems Biology View of Responses to Lignin Biosynthesis Perturbations in Arabidopsis[W] , 2012, Plant Cell.