Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass

Abstract

Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180°C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300-400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.ignocellulosic biomass is an abundant, domestic, renewable feedstock source that can be converted to liquid transportation fuels and other chemicals by fermentation. Cellulosic ethanol is a promising near-term technological option to reduce transportation sector greenhouse gas emissions (1). Because lignocellulosic biomass is made up of the complex structures of cellulose, hemicellulose, and lignin, such feedstock is highly recalcitrant to bioconversion of its carbohydrates into ethanol compared with starch (2, 3). Current biomass fermentation processes for fuels and chemicals have a relatively high cost primarily because of this recalcitrance, which in turn has limited commercialization of biomass ethanol (4). To achieve sustainable energy production, it is necessary to overcome the chemical and structural properties of biomass that inhibit its deconstruction in dedicated bioenergy crops (5).The conversion of lignocellulosic biomass to ethanol is a threestep process that involves pretreatment followed by polysaccharide hydrolysis to simple sugars followed by sugar fermentation to ethanol (6). The presence of lignin in cell walls negatively impacts these conversion steps (7,8). Examination of natural variation in alfalfa, switchgrass, canarygrass, and sorghum has shown that decreased lignin levels improve in vitro enzyme hydrolysis (9, 10). Lignin pathway modification in alfalfa generated transgenic lines with increased enzymati...

Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2‐year comparative analysis of field‐grown lines modified for target gene or genetic element expression

“…For each gene, two transgenic lines, produced from independent transformation events, were designated with suffix V1 and V2. For the GAUT4, FPGS and MYB4 lines, the parental nontransgenic control plants were clones of the plant that served as a source of tissue (explant) for transformation of the specific gene under study (the term ‘clone’ here refers to cuttings such as tillers that are then put in soil and whole plants obtained); for the COMT line, the control plants were clones derived from a line that lost the transgene through segregation in the T1 generation after cross‐hybridization of a transgenic plant with a nontransgenic ‘Alamo’ (Fu et al ., 2011); and for the miRNA line, the control plants were clones from a plant regenerated from the ‘Alamo’ seed‐derived explant source used to produce the miRNA line. In this study, all controls were denoted with the suffix CT.…”

“…Field‐grown switchgrass transgenic lines with high sugar release or good growth phenotypes included those silencing or OE: (i) GAUT4, GAUT4 ‐knockdown (KD) lines down‐regulating expression of galacturonosyltransferase4 , a gene encoding an enzyme involved in pectin biosynthesis (Biswal et al ., Biswal, A.K., Atmodjo, M.A., Li, M., Yoo, C.G., Pu, Y., Lee, Y.‐C., Zhang, J.Y., Bray, A., King, Z., LaFayette, P., Mohanty, S.S., Ryno, D., Yee, K., Thompson, O.A., Rodriguez Jr, M., Winkeler, K., Collins, C., Yang, X., Tan, L., Sykes, R.W., Gjersing, E., Ziebell, A., Turner, G.B., Decker, S.R., Parrot, W., Udvardi, M.K., Mielenz, J., Davis, M.F., Nelson, R.S., Ragauskas, A.J., and Mohnen, D.); (ii) miRNA, miRNA156 ‐overexpression (OE) lines OE miRNA156, a regulator of plant developmental processes (Fu et al ., 2012); (iii) MYB4, MYB4 ‐OE lines OE PvMYB4, an R2R3‐type MYB repressor of the lignin biosynthetic pathway (Shen et al ., 2012, 2013); (iv) COMT, COMT ‐KD lines down‐regulating expression of caffeic acid O ‐methyltransferase, a lignin biosynthetic gene (Fu et al ., 2011); and (v) FPGS, FPGS ‐KD lines down‐regulating expression of folylpolyglutamate synthase 1 , a gene encoding a C1 metabolism enzyme believed to provide methyl groups for lignin biosynthesis (Srivastava et al ., 2015). …”

Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population

“…Forward genetic screens have also led to the discovery of visual and lignin-related phenotypes, such as brown-midrib maize (Zea mays) and sorghum (Sorghum bicolor), which show improved digestion by ruminant animals and increased sugar yield by enzyme hydrolysis (Cherney et al, 1991;Marita et al, 2003;Vermerris et al, 2007). However, reducing lignin content either in mutant or transgenic lines can result in reduced biomass yields (Li and Chapple, 2010;Simmons et al, 2010;Fu et al, 2011aFu et al, , 2011b. This strategy is also undesirable for pyrolytic or chemical catalytic conversion processes that may utilize lignin for hydrocarbon fuels and aromatic coproducts (Venkatakrishnan et al, 2014).…”

Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion

“…In order to reduce these cost elements, researchers have focused on genetic engineering of plants for desirable lignin content or composition by partial removal or redistribution of lignin. The objective of such structural reengineering is to loosen the rigid cell wall structure and expose cellulose and hemicelluloses for enhanced saccharification [11,12,[16][17][18][19][20]. However, lignin genetic engineering generally results in detrimental effects on plant growth and development, and such approaches do not exclude the need for exogenous enzyme addition prior to fermentation [13,20].…”