Precision breeding for RNAi suppression of a major 4-coumarate:coenzyme A ligase gene improves cell wall saccharification from field grown sugarcane

Abstract

Sugarcane (Saccharum spp. hybrids) is a major feedstock for commercial bioethanol production. The recent integration of conversion technologies that utilize lignocellulosic sugarcane residues as well as sucrose from stem internodes has elevated bioethanol yields. RNAi suppression of lignin biosynthetic enzymes is a successful strategy to improve the saccharification of lignocellulosic biomass. 4-coumarate:coenzyme A ligase (4CL) is a key enzyme in the biosynthesis of phenylpropanoid metabolites, such as lignin and flavonoids. Identifying a major 4CL involved in lignin biosynthesis among multiple isoforms with functional divergence is key to manipulate lignin biosynthesis. In this study, two full length 4CL genes (Sh4CL1 and Sh4CL2) were isolated and characterized in sugarcane. Phylogenetic, expression and RNA interference (RNAi) analysis confirmed that Sh4CL1 is a major lignin biosynthetic gene. An intragenic precision breeding strategy may facilitate the regulatory approval of the genetically improved events and was used for RNAi suppression of Sh4CL1. Both, the RNAi inducing cassette and the expression cassette for the mutated ALS selection marker consisted entirely of DNA sequences from sugarcane or the sexually compatible species Sorghum bicolor. Field grown sugarcane with intragenic RNAi suppression of Sh4CL1 resulted in reduction of the total lignin content by up to 16.5 % along with altered monolignol ratios without reduction in biomass yield. Mature, field grown, intragenic sugarcane events displayed 52-76 % improved saccharification efficiency of lignocellulosic biomass compared to wild type (WT) controls. This demonstrates for the first time that an intragenic approach can add significant value to lignocellulosic feedstocks for biofuel and biochemical production.

Sugarcane biomass conversion influenced by lignin

“…Six of the eight evaluated intragenic and transgenic lines did not differ significantly in dry biomass yield from WT, but two lines displayed significantly reduced dry biomass compared to WT. Juice volume per g of fresh stem samples in intragenic and transgenic lines presented a volume of 2–18% higher than WT, although this was not statistically significant …”

“…Juice volume per g of fresh stem samples in intragenic and transgenic lines presented a volume of 2-18% higher than WT, although this was not statistically significant. 38 The efficiency and maximization of the conversion of sugarcane bagasse to bioethanol requires improvements in cultivar production, not only in biomass yield per planted area and fiber content but also in better degradability of biomass during pretreatment and enzymatic digestion. The conversion can be facilitated if the feedstock genotype and composition are optimized for this purpose.…”

Lignin engineering to improve saccharification and digestibility in grasses

“…Sugarcane had reduced height but produced more tillers so biomass was maintained in most lines, even in the field [17]. Saccharification efficiency after dilute acid pretreatment was 52-76% improved in 4CL-suppressed switchgrass and field-grown sugarcane [15,17]. Thus it appears that 4CL can be an effective target for moderate reduction of lignin content with significant benefits for digestibility even in field-grown plants.…”

“…Biomass yield was normal in most cases although rice plants were shorter and less fertile than controls [16]. Sugarcane had reduced height but produced more tillers so biomass was maintained in most lines, even in the field [17]. Saccharification efficiency after dilute acid pretreatment was 52-76% improved in 4CL-suppressed switchgrass and field-grown sugarcane [15,17].…”

“…Suppression of class I 4CLs (i.e., 4CLs potentially involved in monolignol biosynthesis) in switchgrass, rice, sugarcane and sorghum bmr2 mutants leads to around 17-20% lignin reduction, usually with brown midrib-like phenotypes [15][16][17][18]. In cases in which lignin monomer ratios were determined, these generally showed equivalent reductions in G and S, although switchgrass showed differential effects on the three monolignols.…”