BackgroundC-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 plantCleome 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 developingC. hasslerianaseed coats to suggest strategies for engineering C-lignin in a heterologous system, using hairy roots of the model legumeMedicago truncatula.ResultsWe systematically tested strategies for C-lignin engineering using a combination of gene overexpression and RNAi-mediated knockdown in thecaffeic 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 theSelaginella moellendorffiiferulate 5-hydroxylase (SmF5H) gene incomtmutant hairy roots resulted in lines that unexpectedly accumulated high levels of S-lignin.ConclusionC-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 inM. truncatulahairy roots. Cell wall fractionation studies suggested that the engineered C-units are not present in a heteropolymer with the bulk of the G-lignin.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-023-02339-7.