Precision breeding for RNAi suppression of a major 4-coumarate:coenzyme A ligase gene improves cell wall saccharification from field grown sugarcane
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.
Keywords4-coumarate:coenzyme A ligase (4CL) Lignin Intragenic RNAi Sugarcane
The information, data or work presented herein were funded in part by Syngenta. The authors would like to thank Sun Gro Horticulture, Apopka, FL, for donation of Fafard #2 potting mix.
FA conceived the experiments. FA, JHJ, BK and GMM designed the experiments. JHJ isolated candidate genes, evaluated their expression profile and constructed the plasmids. JHJ and HD generated transgenic plants and completed molecular analysis of transgenic plants, BK carried out the field trial including phenotypic, sugar and biomass yield evaluation, monitoring of plots and preparing reports for the regulatory agency. JHJ analyzed cell wall composition. GMM carried out the cell wall saccharification analysis, JHJ and FA wrote the manuscript. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors have no conflict of interest to declare.
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