Charting the Path to Optimize Polysaccharide Accumulation in Bioenergy Sorghum
Authors:
Sang-Jin Kim1,2* ([email protected]), Starla Zemelis-Durfee1, Brian Mckinley1,4, Rylee Sokoloski1, William Aufdemberge1, John Mullet1,4, Federica Brandizzi1,2,3, Timothy J. Donohue5
Institutions:
1Great Lakes Bioenergy Research Center, Michigan State University–East Lansing; 2MSU-DOE Plant Research Laboratory, Michigan State University–East Lansing; 3Department of Plant Biology, Michigan State University–East Lansing; 4Department of Biochemistry and Biophysics, Texas AandM University–College Station; 5University of Wisconsin–Madison
URLs:
Goals
Engineering plants with polysaccharides that can be easily convertible to bioproducts and specialty biofuels.
Abstract
Plants leverage sugars for various essential functions, including energy production, building cells and organs, and transmitting signals. Notably, a significant portion of sugars is channeled towards building the cell wall, a predominant component of plant biomass for bioenergy applications. Mixed-linkage glucan (MLG), a vital cell wall constituent in grasses, has emerged as a promising polysaccharide in bioenergy-related applications due to its abundance in easily fermentable glucans and positive impact on plant biomass digestibility. The team’s work demonstrated that the abundance of MLG is controlled through development along with cell-type specificity by the action of both MLG synthase and hydrolases. This unique modulation of sugar levels in cells through cell wall polysaccharides appears distinctive to MLG and sets it apart from other cell wall polysaccharides in vegetative tissues. Therefore, researchers have been working to improve the plant biomass by storing more MLG in the cell wall that can be easily converted into readily available forms of sugar and increase cell wall digestibility.
Due to its favorable attributes as a biofuel feedstock, such as high biomass yield, resilience to adverse environmental conditions, and low resource requirements, researchers focused on sorghum for the manipulation of MLG biosynthesis. The team demonstrated that overexpression of the major MLG synthase, CSLF6, resulted in significant MLG production and accumulation in transgenic sorghum (CSLF6OX), both in the greenhouse and field conditions. However, the team also observed a developmental degradation of MLG in CSLF6OX sorghum. To mitigate this degradation phenomenon, researchers embarked on the manipulation of the MLG degradation pathway. This strategy involved the identification and characterization of MLG hydrolases, also known as lichenases, with a particular focus on the major lichenase enzymes in sorghum. Using bioinformatics tools, researchers identified three sorghum lichenase candidates that possess a signal peptide for cell wall secretion and a GH17 domain indicative of hydrolase activity as well as high sequence similarity to known lichenases in diverse species. Using synthetic substrates and natural flours, the team optimized the experimental conditions and established the activities of three putative sorghum lichenases. Subsequently, researchers performed a multifaceted approach, including qRT-PCR, RNAseq, in situ hybridization, and concurrent MLG and starch quantification throughout different stages of sorghum leaf development in diurnal conditions. The results revealed cell-type specific, organ- specific, and development-dependent regulation of MLG levels. This regulation is achieved through precise control of CSLF6 and lichenase enzyme levels in different cell types. As a result, the team identified SbLCH1 as the predominant lichenase enzyme in sorghum. Currently, researchers are in the process of generating a knockout line of the major SbLCH1 that researchers identified, using the CRISPER-Cas9 gene-editing technology. The team’s ultimate goal is to integrate a SbLCH1 knockout line with the CSLF6OX hybrid energy sorghum, which researchers have recently developed. By employing this integrated approach, the team anticipates facilitating MLG accumulation by preventing lichenase-dependent degradation, thereby enhancing the accumulation of easily extractable polysaccharides suitable for biofuel production.
References
Kim, S. J., et al. 2023.” Cell- and Development-Specific Degradation Controls the Levels of Mixed-Linkage Glucan in Sorghum Leaves,” Plant Journal 116(2), 360–74. DOI:10.1111/tpj.16376.
Funding Information
This material is based upon work supported by the Great Lakes Bioenergy Research Center, DOE, Office of Science, BER program under Award Number DE-SC0018409.