Systematic approaches to C-lignin engineering in Medicago truncatula

• Published in: Biotechnology for biofuels 2023-06, Vol.16 (1), p.100-100, Article 100
• Main Authors: Ha, Chan Man, Escamilla-Trevino, Luis, Zhuo, Chunliu, Pu, Yunqiao, Bryant, Nathan, Ragauskas, Arthur J, Xiao, Xirong, Li, Ying, Chen, Fang, Dixon, Richard A
• Format: Article
• Language: English
• Subjects: 09 BIOMASS FUELS; Accumulation; Acids; Alcohol; Alcohol dehydrogenase; Alfalfa; Beans; Biomass energy; Bioprocessing; Biosynthesis; C-lignin; Cacti; Caffeic acid; Caffeic acid O-methyltransferase; Cell walls; Chromatography; Cinnamyl-alcohol dehydrogenase; Cleome hassleriana; Cloning; Co-product; DNA methylation; Down-regulation; Energy crops; Engineering; Fractionation; Genetic engineering; Genetically modified plants; Growth; Hairy root; Hairy roots; Legumes; Lignin; Lignocellulose; Medicago truncatula; Metabolic engineering; Metabolites; Methods; Methyltransferase; Mimosaceae; Mutants; Ornamental plants; Oxidases; Physical properties; Plant species; Plants (botany); Polymerization; Polymers; Production processes; Reductases; RNA-mediated interference; Roots; Seed coats; Seeds; Substrates; Transcriptomics; Transferases; Transgenic plants
• ISSN: 2731-3654; 2731-3654; 1754-6834
• DOI: 10.1186/s13068-023-02339-7

C-lignin is a homopolymer of caffeyl alcohol present in the seed coats of a variety of plant species including vanilla orchid, various cacti, and the ornamental plant Cleome hassleriana. Because of its unique chemical and physical properties, there is considerable interest in engineering C-lignin into the cell walls of bioenergy crops as a high-value co-product of bioprocessing. We have used information from a transcriptomic analysis of developing C. hassleriana seed coats to suggest strategies for engineering C-lignin in a heterologous system, using hairy roots of the model legume Medicago truncatula. We systematically tested strategies for C-lignin engineering using a combination of gene overexpression and RNAi-mediated knockdown in the caffeic acid/5-hydroxy coniferaldehyde 3/5-O-methyltransferase (comt) mutant background, monitoring the outcomes by analysis of lignin composition and profiling of monolignol pathway metabolites. In all cases, C-lignin accumulation required strong down-regulation of caffeoyl CoA 3-O-methyltransferase (CCoAOMT) paired with loss of function of COMT. Overexpression of the Selaginella moellendorffii ferulate 5-hydroxylase (SmF5H) gene in comt mutant hairy roots resulted in lines that unexpectedly accumulated high levels of S-lignin. C-Lignin accumulation of up to 15% of total lignin in lines with the greatest reduction in CCoAOMT expression required the strong down-regulation of both COMT and CCoAOMT, but did not require expression of a heterologous laccase, cinnamyl alcohol dehydrogenase (CAD) or cinnamoyl CoA reductase (CCR) with preference for 3,4-dihydroxy-substituted substrates in M. truncatula hairy roots. Cell wall fractionation studies suggested that the engineered C-units are not present in a heteropolymer with the bulk of the G-lignin.