title: 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.
authors.affiliation: Agronomy Department, IFAS, University of Florida, PO Box 110500, Gainesville, FL, 32611, USA.|Institute of Life Science and Natural Resources, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, South Korea.
authors.forename: Je Hyeong
authors.initials: JH
authors.lastname: Jung
authors.affiliation: Agronomy Department, IFAS, University of Florida, PO Box 110500, Gainesville, FL, 32611, USA.
authors.forename: Baskaran
authors.initials: B
authors.lastname: Kannan
authors.affiliation: Agronomy Department, IFAS, University of Florida, PO Box 110500, Gainesville, FL, 32611, USA.
authors.forename: Hugo
authors.initials: H
authors.lastname: Dermawan
authors.affiliation: Novozymes North America Inc, Franklinton, NC, 27525, USA.
authors.forename: Geoffrey W
authors.initials: GW
authors.lastname: Moxley
authors.affiliation: Agronomy Department, IFAS, University of Florida, PO Box 110500, Gainesville, FL, 32611, USA. altpeter@ufl.edu.|Plant Molecular and Cellular Biology Program, IFAS, University of Florida, PO Box 110300, Gainesville, FL, 32611, USA. altpeter@ufl.edu.|University of Florida Genetics Institute, PO Box 103610, Gainesville, FL, 32610, USA. altpeter@ufl.edu.
authors.forename: Fredy
authors.identifier: http://orcid.org/0000-0002-0894-4976
authors.initials: F
authors.lastname: Altpeter
mesh_terms: D001947:Breeding; D002473:Cell Wall / Q000201:enzymology / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D034622:RNA Interference; D031786:Saccharum / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
chemical_list: D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase
keywords: 4-coumarate:coenzyme A ligase (4CL); Intragenic; Lignin; RNAi; Sugarcane